E-Cadherin-Deficient Epithelial Cells Are Sensitive to HDAC Inhibitors

Simple Summary Inactivating mutations in the CDH1 gene cause the cancer syndrome hereditary diffuse gastric cancer and are also frequent in sporadic diffuse gastric and lobular breast cancers. This research aimed to test whether cancers with CDH1 mutations have heightened sensitivity to histone deacetylase (HDAC) inhibitors. The impact of these drugs was tested on several gastric and breast preclinical models with and without CDH1 mutations. One HDAC inhibitor, entinostat, showed a strong inhibitory effect on each of the models with CDH1 mutations. This study highlighted the potential beneficial impact of entinostat for the chemoprevention and/or treatment of gastric and breast cancers carrying CDH1 mutations. Abstract Inactivating germline mutations in the CDH1 gene (encoding the E-cadherin protein) are the genetic hallmark of hereditary diffuse gastric cancer (HDGC), and somatic CDH1 mutations are an early event in the development of sporadic diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In this study, histone deacetylase (HDAC) inhibitors were tested for their ability to preferentially inhibit the growth of human cell lines (MCF10A and NCI-N87) and murine organoids lacking CDH1 expression. CDH1−/− breast and gastric cells were more sensitive to the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat than wild-type cells, with an elevated growth inhibition that was, in part, attributable to increased apoptosis. CDH1-null cells were also sensitive to more class-specific HDAC inhibitors, but compared to the pan-inhibitors, these effects were less robust to genetic background. Increased sensitivity to entinostat was also observed in gastric organoids with both Cdh1 and Tp53 deletions. However, the deletion of Tp53 largely abrogated the sensitivity of the Cdh1-null organoids to pracinostat and mocetinostat. Finally, entinostat enhanced Cdh1 expression in heterozygous Cdh1+/− murine organoids. In conclusion, entinostat is a promising drug for the chemoprevention and/or treatment of HDGC and may also be beneficial for the treatment of sporadic CDH1-deficient cancers.


Introduction
Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome defined by germline CDH1 or, occasionally, CTNNA1 variants in either an isolated individual with diffuse gastric cancer (DGC), or in a family with one or more cases [1]. Pathogenic germline CDH1 mutations lead to a lifetime risk of developing DGC and lobular breast cancer (LBC) of up to 70% and 40%, respectively [1]. Tumor formation in HDGC requires inactivation of the retained wild-type CDH1 allele by mechanisms including mutation and promoter hypermethylation [2]. The CDH1 gene encodes E-cadherin, a homophilic cell-to-cell adhesion protein involved in the maintenance of cellular polarity, epithelial A CDH1-null strain of the gastric cancer cell line NCI-N87 (ATCC, CRL-5822) was generated using CRISPR-Cas9 [9]. Briefly, the sequence 5 -ATTCACATCCAGCACATCCA-3 was used to target exon 10 of CDH1 and induce a single base frameshift, followed by a stop codon. Isogenic NCI-N87 cells were cultured in DMEM/F12/Glutamax with 10% fetal bovine serum (10091148, Thermo Fisher Scientific, Waltham, MA, USA). Both cell lines were grown at 37 • C with 5% CO 2 .

Drugging Assay on Cell Culture
For drugging experiments, cells were seeded in a 96-well plate at 4000 or 10,000 cells/well for MCF10A and NCI-N87, respectively, for 48 h drugging (2500 and 6500 cells for 72 h experiment). On day one, nuclei on the outer well were counted by using 1 µg/mL Hoechst (H1399, Thermo Fisher Scientific, Waltham, MA, USA) on a Cytation imager (Biotek, Winooski, VT, USA), with the associated Gen5 software (Biotek, Winooski, VT, USA). If the ratio of WT vs. CDH1 −/− cells was between 0.6 and 1.4, cells were processed for drugging. The different drugs used (Table S1) were solubilized in DMSO and diluted in the appropriate media. Only DMSO was added to the control wells to a concentration of 0.1 to 0.4% to match the concentration in the drugging wells. After 48 or 72 h, cells were stained by using 0.25% PFA, 0.075% Saponin and 1 µg/mL Hoechst in 1× PBS. Cell count was assessed by using Cytation 5 imager and normalized to the DMSO controls. Drugging was performed as technical triplicates for every concentration and combined.
Organoids were cultured in organoid complete media (Tables S2 and S3). Gastric organoids were passaged as single cells every 5-6 days and seeded at a concentration of 20 cells/µL of Matrigel. Mammary organoids were passaged every 7 days and seeded at a concentration of 60 cells/µL of Matrigel. Organoids were grown at 37 • C with 5% CO 2 .

Drugging Assay on Organoids
For drugging experiments, 250 (gastric) or 300 (mammary) cells/well were seeded in a 384-well plate (day 0). Organoids were induced with 0.5 µg/µL of endoxifen (E8284, Sigma-Aldrich, St. Louis, MO, USA) at day 1 for the gastric organoids and day 2 for the mammary organoids. The expression of tdTomato was then assessed to check for the proper induction of the organoids that were then processed for drugging at day 2 for the gastric organoids and day 4 for the mammary organoids. WT, Cdh1 −/− and Cdh1 −/− /Tp53 −/− organoids were seeded as technical triplicates per drug concentration. After 48 h, pictures of the wells were taken, using Z-stack on a Cytation 5 imager (Biotek, Winooski, VT, USA). Pictures were then combined and focus-stacked, using a custom Python script. The area of the organoids was measured, using ImageJ, and normalized to the DMSO controls. The circularity of the Cdh1 +/− organoids was measured by using ImageJ shape descriptor, with a value of 1 indicating a perfect circle (circularity = 4π(area/perimeterˆ2).
FACS was performed on WT and Cdh1 +/− organoids after 48 h of drugging with entinostat, to assess for E-cadherin expression. Briefly, organoids were harvested and processed into single cells before fixation in 4% PFA for 15 min. Cells were then resuspended in 2% BSA-PBS containing the E-cadherin Alexa Fluor 647-conjugated antibody 1:200 (FAB7481R, R&D Systems, Minneapolis, MN, USA) for 30 min on ice. Cells were then washed in PBS and resuspended in 1% BSA-PBS. Samples were analyzed by using a BD Fortessa Flow cytometer.

Statistical Analysis
All data are presented as the mean ± SEM. Analyses were performed by using Prism 8 software. Gaussian distribution was assessed by using the Shapiro-Wilk test, and the comparisons between two groups were performed by using multiple t-tests. Comparisons between WT, Cdh1 −/− and Cdh1 −/− /Tp53 −/− organoids were performed by using 2-way ANOVA with Dunnett or Tukey multiple comparisons test. Correlation analysis between CDH1 expression and HDACs were performed, using Spearman correlation followed by a Holm-Bonferroni test (adjusted p-value). Statistically significant results are labeled as * p < 0.05, ** p < 0.01 and *** p < 0.001.

HDAC Classes Show Contrasting Correlations with CDH1 Expression
To better understand the relationship between the different HDAC classes and CDH1 expression in gastric cancer, we performed hierarchical clustering on gene-expression data from 415 gastric cancers (TCGA and STAD dataset). Notably, when comparing the zincdependent HDACs, the Class I and IIb HDACs showed an overall inverse relationship to the Class IIa and IV HDACs ( Figure 1A). When compared directly to CDH1 expression ( Figure 1B-F and Spearman correlation Table S4), each of the Class IIa HDACs was significantly negatively correlated with CDH1 expression, with a mean Spearman correlation of −0.23 ( Figure 1C). Of the Class I and IIb HDACs, both HDAC1 and HDAC10 showed a significant positive correlation with CDH1 ( Figure 1B,D). In Class III, SIRT4 was negatively correlated with CDH1 expression, whereas both SIRT5 and SIRT7 were positively correlated ( Figure 1E). Finally, HDAC11 from Class IV was not correlated with CDH1 expression ( Figure 1F). Overall, these results illustrate the complex, but connected, relationships between the different HDAC classes and transcriptional patterns involving CDH1 in gastric cancer. The correlations also suggest that targeting Class IIa HDACs might provide greater specificity for gastric cancers with low CDH1 expression.

HDAC Classes Show Contrasting Correlations with CDH1 Expression
To better understand the relationship between the different HDAC classes and CD expression in gastric cancer, we performed hierarchical clustering on gene-express data from 415 gastric cancers (TCGA and STAD dataset). Notably, when comparing zinc-dependent HDACs, the Class I and IIb HDACs showed an overall inverse relat ship to the Class IIa and IV HDACs ( Figure 1A). When compared directly to CDH1 pression ( Figure 1B-F and Spearman correlation Table S4), each of the Class IIa HDA was significantly negatively correlated with CDH1 expression, with a mean Spearm correlation of −0.23 ( Figure 1C). Of the Class I and IIb HDACs, both HDAC1 and HDA showed a significant positive correlation with CDH1 ( Figure 1B,D). In Class III, SIRT4 negatively correlated with CDH1 expression, whereas both SIRT5 and SIRT7 were p tively correlated ( Figure 1E). Finally, HDAC11 from Class IV was not correlated w CDH1 expression ( Figure 1F). Overall, these results illustrate the complex, but connec relationships between the different HDAC classes and transcriptional patterns involv CDH1 in gastric cancer. The correlations also suggest that targeting Class IIa HDA might provide greater specificity for gastric cancers with low CDH1 expression.  The HDACs with a significant correlation with CDH1 expression are labeled with an asterisk (*). LC = Lauren Classification, representing the different gastric cancer subtypes: red, diffuse; green, intestinal; and blue, mixed subtypes.

Breast and Gastric Cells Deficient for E-Cadherin Are Sensitive to HDAC Inhibitors
To confirm earlier observations of the enhanced inhibitory effect of HDAC inhibitors on E-cadherin-deficient cells [12], we treated an isogenic pair of MCF10A-wild-type (WT) and MCF10A-CDH1 −/− cells with the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat and confirmed the greater inhibition of MCF10A-CDH1 −/− compared to the MCF10A-WT after 48 h of treatment ( Figure S1 and Table S5). The observed inhibitory effects occurred at concentrations at which histone deacetylation inhibition has previously been observed in other cell lines [31,32]. A maximum decrease (with a p-value < 0.05) of 18% in the number of MCF10A-CDH1 −/− cells compared to MCF10A-WT was observed for entinostat, 15% for pracinostat, 14% for mocetinostat and 13% for vorinostat. Other FDA-approved pan-HDAC inhibitors, namely belinostat, panobinostat and romidepsin, did not show any evidence of a synthetic lethal effect on the MCF10A isogenic cells (data not shown).
To confirm earlier observations of the enhanced inhibitory effect of HDAC inhibitors on E-cadherin-deficient cells [12], we treated an isogenic pair of MCF10A-wild-type (WT) and MCF10A-CDH1 −/− cells with the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat and confirmed the greater inhibition of MCF10A-CDH1 −/− compared to the MCF10A-WT after 48 h of treatment ( Figure S1 and Table S5). The observed inhibitory effects occurred at concentrations at which histone deacetylation inhibition has previously been observed in other cell lines [31,32]. A maximum decrease (with a p-value < 0.05) of 18% in the number of MCF10A-CDH1 −/− cells compared to MCF10A-WT was observed for entinostat, 15% for pracinostat, 14% for mocetinostat and 13% for vorinostat. Other FDA-approved pan-HDAC inhibitors, namely belinostat, panobinostat and romidepsin, did not show any evidence of a synthetic lethal effect on the MCF10A isogenic cells (data not shown).

Pan-HDAC Inhibitors Are More Robust to Genetic Background Than More Class-Specific HDAC Inhibitors
In order to identify any HDAC class-specific effects, we tested a panel of HDAC inhibitors with increased specificity (listed in Table S1). These HDAC inhibitors were first tested in the MCF10A isogenic pairs ( Figure S2A In this cell line, the inhibitory effect of a proportion of the targeted HDAC inhibitors was greater than the pan-HDAC inhibitors, with a maximum difference up to 25% for RGFP966, 39% for LMK-235, 27% for TH-34, 32% for SirReal2 and 35% for LC-0296. In contrast to the isogenic MCF10A cells, the targeted HDAC inhibitors showed few synthetic lethal effects when tested on CDH1 isogenic NCI-N87 cells. Romidepsin (HDAC1 and 2 inhibitor) was highly toxic to both NCI-N87 cell lines after 72 h (data not shown), but had a synthetic lethal effect up to 27.5% difference between the NCI-N87-CDH1 −/− and NCI-N87-WT cells after only 24 h of treatment ( Figure S3). Tasquinimod (HDAC4 inhibitor) presented a marginal synthetic lethal effect after 72 h (6%, Figure S3). Finally, Sis17 (HDAC11 inhibitor) also induced a difference in the number of NCI-N87-CDH1 −/− compared to NCI-N87-WT cells (18.6% at 20 µM, Figure S3), but this observed difference was due to stimulation of NCI-N87-WT cell growth, not an increased toxicity for CDH1 −/− cells. Two of the targeted HDAC inhibitors, TH34 (HDAC6, 8 and 10 inhibitor) and LC-0296 (SIRT3 inhibitor), induced a significant "reverse" synthetic lethal effect, with greater toxicity against the WT cells compared to the CDH1 −/− cells after 72 h of treatment ( Figure S3). Together, these results suggest that the simultaneous inhibition of multiple HDACs with pan-HDAC inhibitors is more likely to provide robust inhibition of E-cadherin-deficient cells across different genetic backgrounds than a more targeted approach.

Pan-HDAC Inhibitors Preferentially Induce Apoptosis and Decrease Proliferation in E-Cadherin-Deficient Cells
To assess if the increased sensitivity of CDH1 −/− cells to the pan-HDAC inhibitors was due to a cytotoxic or cytostatic mechanism, we first measured the percentage of total apoptosis by Annexin-V-FITC/propidium iodide flow cytometry. Cells were drugged for 48 h, using the drug concentrations that had the greatest synthetic lethal effect in the NCI-N87 nuclei count assay ( Figure 3A). We observed a significant increase in apoptosis in NCI-N87-CDH1 −/− cells compared to NCI-N87-WT cells for three of the pan-HDAC inhibitors tested, with relative increases of 18% for 2.5 µM entinostat, 25% for 0.3 µM pracinostat and 34% for 2.5 µM vorinostat ( Figure 3B).
The potential cytostatic effect of pan-HDAC inhibitors on the NCI-N87 isogenic cells was also examined by cell-cycle analysis. After 48 h of drugging, pracinostat and vorinostat significantly reduced the percentage of NCI-N87-CDH1 −/− cells in G1 phase compared to NCI-N87-WT cells and increased the proportion of cells in the S phase ( Figure 3D). Entinostat and mocetinostat did not differentially impact the cell cycle in WT and NCI-N87 cells deficient for E-cadherin. Together, the results indicate that the increased sensitivity of CDH1 −/− cells to pan-HDAC inhibitors is due to both cytostatic and cytotoxic effects, but these effects are not consistent between the four drugs tested. Cancers 2022, 14, x FOR PEER REVIEW 9 of 20

The Characterization of E-Cadherin-Deficient Mouse Gastric Organoids
To test the pan-HDAC inhibitors in a more physiologically relevant model, we generated mouse gastric organoids originating from transgenic, endoxifen-inducible Crelox mice under the control of the CD44 stem cell promoter. Each organoid construct included homozygous floxed alleles encoding the red fluorescent marker protein td-Tomato. WT organoids were generated from the stomachs of CD44-Cre/tdTomato (fl/fl) mice. Cdh1 −/− organoids were generated from CD44-Cre/Cdh1 (fl/fl) /tdTomato (fl/fl) mice with loxP sites flanking exons 6 to 10 of Cdh1; double Cdh1/Tp53 knockout organoids were generated from CD44-Cre/Cdh1 (fl/fl) /Tp53 (fl/fl) /tdTomato (fl/fl) mice with loxP sites flanking exons 6 to 10 of Cdh1 and exons 2 to 10 of Tp53. CDH1 and TP53 are the most frequently mutated genes in human DGC, occurring in 33% and 34% of tumors, respectively [33], and knockout of both these genes under the control of the Atp4b promoter has previously been shown to lead to metastatic gastric cancer [34].

Gastric Organoids
Gastric organoids were drugged with pan-HDAC inhibitors for 48 h before their area was measured, and the averages were normalized to their respective DMSO-treated controls ( Figure 5). As previously observed in MCF10A and NCI-N87 cells, entinostat preferentially inhibited the growth of cells lacking E-cadherin, with a decrease in area of up to 44% at 7.5 µM after 48 h of treatment in Cdh1 −/− organoids and 40% in Cdh1 −/− /Tp53 −/− organoids compared to WT ( Figure 5A,E and Table S5). Pracinostat also induced a synthetic lethal effect with a maximum difference at 0.5 µM of 40% in the area of Cdh1 −/− compared to WT organoids ( Figure 5B,E). For the Cdh1 −/− /Tp53 −/− organoids, a significant synthetic lethal effect was only observed at 0.25 µM, with a difference of 20% compared to the WT organoids. Mocetinostat decreased growth of Cdh1 −/− organoids by 39% at 0.63 µM relative to the WT organoids, but no statistically significant synthetic lethal effect was observed for the Cdh1 −/− /Tp53 −/− organoids ( Figure 5C,E). Vorinostat showed no preferential growth inhibition of E-cadherin-negative organoids, regardless of the Tp53 genotype ( Figure 5D,E). Together, these results suggest that Tp53 mutation is a marker of partial resistance to pracinostat and mocetinostat, but not entinostat, which retains a strong synthetic lethal effect in gastric organoids with both Cdh1 −/− and Cdh1 −/− /Tp53 −/− genetic backgrounds.
organoids. Mocetinostat decreased growth of Cdh1 −/− organoids by 39% at 0.63 μM relative to the WT organoids, but no statistically significant synthetic lethal effect was observed for the Cdh1 −/− /Tp53 −/− organoids ( Figure 5C,E). Vorinostat showed no preferential growth inhibition of E-cadherin-negative organoids, regardless of the Tp53 genotype ( Figure  5D,E). Together, these results suggest that Tp53 mutation is a marker of partial resistance to pracinostat and mocetinostat, but not entinostat, which retains a strong synthetic lethal effect in gastric organoids with both Cdh1 −/− and Cdh1 −/− /Tp53 −/− genetic backgrounds. Figure 5. E-cadherin-deficient gastric organoids are more sensitive to pan-HDAC inhibitors. WT, Cdh1 −/− and Cdh1 −/− /Tp53 −/− organoids were drugged for 48 h with the different pan-HDAC inhibitors, and the area of the organoids was then measured and normalized to their respective vehicle controls. RFP fluorescence was used to detect tdTomato and determine the area of the organoids, using

Mammary Organoids
To further confirm the efficacy of the pan-HDAC inhibitors on E-cadherin-deficient cells, mammary organoids from the same CD44-Cre/Cdh1 (fl)/(fl) /tdTomato mice were generated and drugged by following a similar protocol to the gastric organoids [30]. As previously observed in the gastric organoids, entinostat, pracinostat and mocetinostat preferentially reduced the growth of E-cadherin-deficient mammary organoids compared to the WT, with a decrease in area up to 32% for 15 µM entinostat, 35% for 0.25 µM pracinostat and 24% for 10 µM mocetinostat ( Figure 6A-C and Table S5). Similar to the gastric organoids, vorinostat showed little evidence of a Cdh1 synthetic lethal effect on the mammary organoids ( Figure 6D). ated and drugged by following a similar protocol to the gastric organoids [30]. As previously observed in the gastric organoids, entinostat, pracinostat and mocetinostat preferentially reduced the growth of E-cadherin-deficient mammary organoids compared to the WT, with a decrease in area up to 32% for 15 μM entinostat, 35% for 0.25 μM pracinostat and 24% for 10 μM mocetinostat ( Figure 6A-C and Table S5). Similar to the gastric organoids, vorinostat showed little evidence of a Cdh1 synthetic lethal effect on the mammary organoids ( Figure 6D).  Table S5. Statistically significant results are labeled as * p < 0.05 and ** p < 0.01.

Entinostat Promotes E-Cadherin Expression in Organoids Heterozygous for Cdh1 Mutation
The growth-inhibition experiments described above focused on achieving chemoprevention in HDGC by inhibiting the survival of the CDH1-null multifocal stage T1a SRCCs that are present in CDH1 germline mutation carriers. An alternative to this approach is to Figure 6. E-cadherin-deficient mammary organoids are more sensitive to pan-HDAC inhibitors. WT and Cdh1 −/− mammary organoids were generated from the same Cre-lox mice than the gastric organoids and drugged with the pan-HDAC inhibitors. The mammary organoids area were then assessed after 48 h of drugging and normalized to their respective DMSO. (A) Entinostat induced a synthetic lethal effect, with a maximum of 32% differences at 15 µM, between WT and Cdh1 −/− organoids. (B) Pracinostat decreased Cdh1 −/− growth up to 35% at 0.25 µM compared to the WT. (C) Mocetinostat induced an SL effect, with a maximum of 24% decrease at 10 µM, in the Cdh1 −/− area compared to WT mammary organoids. (D) Vorinostat presented an SL effect at the lowest concentration, with a difference of 19% between the Cdh1 −/− and WT organoids; n = 3-5 per compound. All the IC50 are summarized in Table S5. Statistically significant results are labeled as * p < 0.05 and ** p < 0.01.

Entinostat Promotes E-Cadherin Expression in Organoids Heterozygous for Cdh1 Mutation
The growth-inhibition experiments described above focused on achieving chemoprevention in HDGC by inhibiting the survival of the CDH1-null multifocal stage T1a SRCCs that are present in CDH1 germline mutation carriers. An alternative to this approach is to exploit the high frequency with which the second CDH1 allele is inactivated epigenetically in these foci and use drugs to re-express it [2,38]. Re-expression of the epigenetically silenced CDH1 allele would be predicted to restore adherens junctions and associated signaling, preventing the generation of CDH1-null gastric epithelial cells, which can be displaced into the lamina propria, beyond the influence of normal regulatory cues. Pan-HDAC inhibitors, including entinostat, pracinostat, mocetinostat and vorinostat, have previously been shown to activate CDH1 expression [39,40], raising the possibility that these drugs could both eliminate established stage T1a SRCCs and prevent their formation.
We had previously observed that Cdh1 +/− heterozygote gastric organoids generated from CD44-Cre/Cdh1 (fl/WT) /tdTomato mice showed similar morphology to gastric organoids developed from CD44-Cre/Cdh1 (fl/fl) /tdTomato mice in which both Cdh1 alleles had been inactivated ( Figure 7A). Immunofluorescence with anti-E-cadherin antibodies confirmed that a high proportion of cells in the Cdh1 heterozygous organoids were Ecadherin-null ( Figure 7B), presumably due to the epigenetic silencing of the wild-type Cdh1 allele during the development of the organoids, similar to the natural history of HDGCs stage T1a gastric SRCCs [2]. To test the effect of entinostat on these Cdh1 heterozygote organoids, Cdh1 +/− organoids were treated with entinostat or DMSO control 24 h after endoxifen induction. Forty-eight hours later, strong tdTomato expression was observed in these Cdh1 +/− organoids, demonstrating the activation of the Cre recombinase ( Figure 7B). The DMSO-treated organoids displayed a disrupted shape and the presence of evaginations typical of Cdh1-null organoids. In contrast, entinostat treatment prevented the formation of evaginations and the organoids maintained the circular shape characteristic of WT organoids. Our analysis of the morphology of these organoids confirmed an increase circularity (characterized by a value of 1) with entinostat compared to DMSO ( Figure 7C). Immunofluorescence demonstrated that E-cadherin was strongly expressed in the entinostat-treated Cdh1 +/− organoids compared to the same organoids treated with DMSO ( Figure 7B). To confirm this effect, the expression of E-cadherin was verified by flow cytometry in WT and Cdh1 +/− organoids after drugging ( Figure 7D). The E-cadherin level was unchanged in the WT organoids following entinostat treatment, as compared to DMSO (fold change = 0.94, p-value = 0.8). However, E-cadherin expression was significantly increased with entinostat in the Cdh1 +/− organoids (fold change of 1.60, p-value < 0.001). Together, these results demonstrate that entinostat is increasing the expression of the WT allele in heterozygous Cdh1 +/− organoids, leading to normalized organoid morphology.

Discussion
By virtue of their central role in transcriptional regulation, it is unsurprising that the expression of HDACs is dysregulated in cancer, including breast and gastric tumors [41]. However, we were interested in understanding if any HDACs were co-regulated with CDH1 in gastric cancer. We first examined the correlation between individual HDACs and CDH1 expression in gastric tumors. Of the 18 HDACs, half of them were significantly Entinostat increases E-cadherin expression in Cdh1 +/− organoids, leading to rounded morphology. Cdh1 +/− organoids were induced at day 1 followed by drugging at day2 with entinostat or DMSO for 48 h. (A) Bright field and tdTomato expression were assessed at day 4 in WT, Cdh1 +/− and Cdh1 −/− organoids. Cdh1 decrease of expression was associated with a loss of circularity and the presence of evagination. (B) Immunofluorescence was performed on Cdh1 +/− organoids following 48 h of treatment with entinostat or its respective DMSO concentration. Bright field and tdTomato showed a maintenance of a circular morphology following entinostat treatment. Immunofluorescence on Cdh1 +/− organoids was performed with E-cadherin stained in green, tdTomato in red and DAPI in blue for nuclear staining. A decrease of E-cadherin was observed after induction in the Cdh1 +/− DMSO-treated organoids, whereas entinostat treated Cdh1 +/− organoids maintained a strong E-cadherin expression. (C) Circularity of the Cdh1 +/− organoids was measured after DMSO or entinostat drugging, with a value of 1 representing a perfect circle (n = 43-97 organoids analyzed). (D) E-cadherin expression after entinostat drugging was measured by flow cytometry for WT and Cdh1 +/− gastric organoids and normalized to their respective DMSO (n = 4). Entinostat treatment increased the expression of E-cadherin in the Cdh1 +/− organoids. Statistically significant results are labeled as * p < 0.05 and *** p < 0.001.

Discussion
By virtue of their central role in transcriptional regulation, it is unsurprising that the expression of HDACs is dysregulated in cancer, including breast and gastric tumors [41]. However, we were interested in understanding if any HDACs were co-regulated with CDH1 in gastric cancer. We first examined the correlation between individual HDACs and CDH1 expression in gastric tumors. Of the 18 HDACs, half of them were significantly correlated with CDH1 expression, including four HDACs that were positively correlated (HDAC1, HDAC10, SIRT5 and SIRT7) and five that were negatively correlated (HDAC4, HDAC5, HDAC7, HDAC9 and SIRT4). These correlations may reflect the key role E-cadherin plays in major transcriptional patterns, particularly in the epithelial-mesenchymal transition [35].
HDAC inhibitors exert their anticancer effects through a range of cytotoxic or cytostatic mechanisms that are likely to be context dependent, including cell-cycle arrest, apoptosis and promotion of a terminally differentiated state [17,42]. We observed strong pro-apoptotic effects for each of entinostat, pracinostat, mocetinostat and vorinostat in the NCI-N87 cell lines. Notably, the percentage of cells that were apoptotic was higher in the NCI-N87-CDH1 −/− line compared to WT cells, following treatment with each drug, suggesting that the increased sensitivity of CDH1-deficient cells to HDAC inhibitors is mediated, at least in part, by increased cytoxicity of the HDAC inhibitors in these cells. The four HDAC inhibitors also had a cytostatic effect on the isogenic NCI-N87 cells, but it was less pronounced than the pro-apoptotic effect. However, as observed with the apoptotic markers, the inhibitory effect was amplified in the CDH1-deficient cells, even though this increase was only significant for vorinostat and mocetinostat. Although other mechanisms such as autophagic apoptosis [25] or ferroptosis [25,43] may also be involved, these results show that the pan-HDAC inhibitors are acting preferentially on CDH1-deficient cells through both cytotoxic and cytostatic mechanisms, as also observed in other cell lines [27,44,45].
To validate the effects of the pan-HDAC inhibitors in a more complex gastric model, entinostat, pracinostat, mocetinostat and vorinostat were tested on inducible mouse-derived gastric organoids with either WT, Cdh1 −/− or Cdh1 −/− /Tp53 −/− genetic backgrounds. Organoid models better reproduce the epithelial cell's local environment and drug responses correlate well with in vivo effects [46]. Immunofluorescence on these organoids showed that the loss of Cdh1 was associated with a disrupted morphology, evaginations and higher proliferation than control organoids. The addition of a Tp53 mutation aggravated the irregular shape and the number of cells displaced from the organoid following loss of Cdh1 expression. These results were consistent with previous studies that showed that E-cadherin and Tp53 inactivation are associated with an increase in proliferation, migration and invasiveness of cells [35,47].
Of the four pan-HDAC inhibitors tested, each of entinostat, pracinostat and mocetinostat exhibited significant synthetic lethal effects in the E-cadherin-deficient gastric and mammary gland organoids compared to WT. However, the additional deletion of Tp53, the most commonly mutated gene in DGC, in the gastric organoids strongly reduced this effect with pracinostat and mocetinostat, but not entinostat. Notably, entinostat has generally been well tolerated in clinical trials, with most side effects being mild to moderate and manageable with supportive care [48]. The differential inhibition between wild-type and CDH1-null cells was frequently statistically significant at sub-micromolar concentrations and consistent with the effective concentrations observed in numerous cancer cell lines [17,48,49].
Before testing entinostat in a chemoprevention clinical trial, it will be necessary to first demonstrate efficacy in an animal model. We recently developed a novel mouse model with tamoxifen-inducible inactivation of Cdh1 and Tp53 that develops diffuse type gastric cancers in~3 months (unpublished data). Candidate chemoprevention drugs could be tested in this model in mice with established cancers, but also in others which would be treated with drug 2-4 weeks after tamoxifen-induction, with the tumor burden being quantified a further 8-10 weeks later.
Chemoprevention of HDGC can be achieved by either targeting the early stage T1a SRCC foci before they have acquired the capacity for invasion beyond the muscularis mucosae, or by reducing the risk of epigenetic silencing of the second CDH1 allele, leading to complete loss of E-cadherin expression and migration of epithelial cells into the lamina propria. Our data suggest that entinostat, in contrast with other potential chemoprevention drugs [9,10], may be able to reduce cancer risk by both promoting apoptosis in established CDH1-null foci and preventing the initiation of new foci by maintaining the integrity of the epithelial plane.
We predict that the sub-millimeter median size of the HDGC stage T1a SRCC foci [50] and their genetic homogeneity (i.e., only CDH1 mutations) will allow the use of chemoprevention drug doses that are well tolerated. Moreover, we predict that the relative indolence of these early cancers will enable treatment schedules that minimize the risk of accumulated toxicities and, at the same time, make compliance more likely (e.g., repeated treatment every 1-2 years). The small size and indolence of the HDGC stage T1a foci sets these cancers apart from advanced highly mutated cancers, which require large pharmacological windows and aggressive treatment strategies. Reductions in drug dose may also be enabled by the identification of synergistic combinations between entinostat and other synthetic lethal drugs. Methods to deliver drugs directly to the gastric mucosa would also be anticipated to minimize many of the side effects associated with systemic treatment. Ideally, drugs could be delivered to the mucosa at the same time as annual surveillance endoscopy. Our data showing drug efficacy in breast MCF10A isogenic cells and mouse mammary organoids also suggest that CDH1 synthetic lethal drugs, such as entinostat, may also be effective in reducing the risk of LBC and lobular carcinoma in situ (LCIS) in HDGC families. Moreover, given the high frequency of somatic CDH1 mutations in sporadic DGC, LCIS and LBC and their association with poor prognosis [51,52], these drugs may also be useful for the treatment of sporadic cancers.
The molecular vulnerability that sensitizes CDH1-null cells to pan-HDAC inhibitors is unclear. Although the effect may be related to a specific histone or non-histone target, the maintenance of a synthetic lethal effect with multiple HDAC inhibitors, both specific and pan-inhibitors, suggests that their effect may be more general, reflecting a shift in the balance of inputs into major cell survival pathways in CDH1-null cells, such as the PI3K/AKT, RAS and MAPK signaling pathways.
In conclusion, our study has demonstrated that E-cadherin-deficient gastric and breast cells are more sensitive to HDAC inhibitors than their wild-type isogenic pairs and suggests that that pan-HDAC inhibitors-in particular, entinostat-may have a role in the chemoprevention of HDGC and the treatment of sporadic LBC and DGC.

Conclusions
We have determined that pan-HDAC inhibitors preferentially target cells lacking E-cadherin expression in breast cells, gastric cancer cells and mouse-derived gastric and mammary organoids. Of all the pan-HDAC inhibitors tested, entinostat showed the most promising results, with a synthetic lethal effect in all our models. These results showed that pan-HDAC inhibitors could lead to new a chemoprevention and/or treatment strategy for HDGC patients with CDH1 mutations.
Supplementary Materials: The following supporting information can be downloaded online at https://www.mdpi.com/article/10.3390/cancers14010175/s1. Figure S1: MCF10A E-cadherindeficient cells are more sensitive to pan-HDAC inhibitors than normal MCF10A. Figure S2: Specific HDAC inhibitors induce a synthetic lethal effect in MCF10A deficient for E-cadherin. Figure S3: Specific HDAC inhibitors have less effect on NCI-N87 cells compared to pan-HDAC inhibitors. Figure S4: E-cadherin and Tp53 expression in WT and E-cadherin-deficient organoids. Table S1: List of the different compounds used for the drugging experiment, along with their respective targets. Table S2: Gastric organoid complete media. Table S3: Mammary organoid complete media. Table S4: Adjusted p-value of the Spearman correlation between HDACs and CDH1 expression. Table S5: IC50 of the different pan-HDAC inhibitors obtained in our 2D and 3D models.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.